Prevalence of extramucosal fungal elements in sinonasal polyposis: a mycological and pathologic study in an Egyptian population

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1 Available online at American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) Prevalence of extramucosal fungal elements in sinonasal polyposis: a mycological and pathologic study in an Egyptian population Ahmed Bassiouny, MD a, Ashraf Ragab, MD a, Abdel Fattah Attia, MD b, Ahmed Atef, MD a,, Nadia Hafez, MD b, Essam Ayad, MD c, Hany Sameer, MD a a Faculty of Medicine, Department of Otolaryngology, Cairo University, Cairo, Egypt b Faculty of Medicine, Department of Microbiology, Cairo University, Cairo, Egypt c Faculty of Medicine, Department of Pathology, Cairo University, Cairo, Egypt Received 4 February 2010 Abstract Objective and hypothesis: The objective of the study was to define the true incidence of fungal elements in the nasal and sinus mucous in cases of chronic rhinosinusitis (CRS) with bilateral polyposis compared with normal controls in an Egyptian African population via mycological and histologic techniques. Study design: This study was conducted prospectively on 100 patients with the clinical diagnosis of CRS with bilateral nasal polyposis. Fifty volunteers with no history of nasal or paranasal sinus disease served as a control group. Results and conclusion: The postulated criteria for the diagnosis of allergic fungal sinusitis were present in 92% of CRS with polyposis, suggesting that fungi are involved in the disease process of most CRS patients Elsevier Inc. All rights reserved. 1. Introduction The etiology of nasal polyposis was a subject of great conflict between the authors; some of the most commonly reported causes of nasal polyps include infection, allergy, immunologic factors, metabolic diseases, hereditary diseases, and autonomic dysfunction [1]. A new etiologic factor was introduced by Safirstein in 1976 who noticed the combination of nasal polyposis, thick tenacious mucin, crust formation, and sinus yielding Aspergillus; and this started the era of fungal condemnation and gave rise to several descriptive nomenclatures such as allergic aspergillosis of the paranasal sinus [2], allergic aspergillus sinusitis [3], and allergic fungal rhinosinusitis [4]. In 1999, Ponikau et al [5] presented their Mayo clinic experience that showed that most patients with chronic Corresponding author. Faculty of Medicine, Department of Otolaryngology, Cairo University, 7 El Shaheed Mahmoud Afifi St, Of El Nozha St, Almaza, Cairo, Egypt. address: amratef@dr.com (A. Atef). rhinosinusitis (CRS) actually meet their verified criteria for allergic fungal sinusitis (AFS); and they stated a new theory for the pathogenesis that gave rise to the term eosinophilic fungal rhinosinusitis (EFS). This new theory was presented during the 1999 fall meeting of the American Rhinologic Society (New Orleans); and it was discussed widely during the XVIIth World Congress of the International Federation of Oto-Rhino- Laryngological Societies held in Cairo in 2002, although this theory did not gain the acceptance of all the polyposisminded rhinologists. However, it provided a new hope for those who are suffering from this dilemma. The aim of this work is to define the true incidence of fungal elements in the nasal and sinus mucous in cases of CRS with bilateral polyposis compared with normal controls in an Egyptian African population via mycological and histologic identification of the various fungal species colonizing the viscid allergic mucin using recent handling techniques in collecting the mucin, and to compare the fungal yield quantitatively and qualitatively with the 2 other reports in the literature studying the fungi in American and European population /$ see front matter 2011 Elsevier Inc. All rights reserved. doi: /j.amjoto

2 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) Material and methods 2.1. Patients and controls Inclusion criteria This study was conducted on 100 patients with the clinical diagnosis of CRS with bilateral nasal polyposis. Fifty volunteers with no history of nasal or paranasal sinus disease, with no symptoms of inhalant allergy, with no history of smoking, and with normal-appearing mucosa confirmed by nasal endoscopy served as a control group. Patients and controls were instructed not to use any steroidal medications 1 month before the study to prevent diminishing the eosinophilic inflammation Exclusion criteria All patients with history of diabetes mellitus or with local or systemic corticosteroids medication 1 month before the study were excluded from this study. All patients studied presented to the Kasr El Aini hospital outpatient clinic during the period from August 2002 to July Their ages ranged between 14 and 56 years. The study included 66 males and 34 females, and all were subjected to the following: Full ENT examination. Nasal endoscopic examination. Computed tomographic scanning of the nose and paranasal sinuses (coronal and axial 5-mm thick cuts) in both soft tissue and bone windows. Informed consents were signed by all the patients for the surgery and for sharing in the scientific research, and the study design was approved by the Cairo University Ethics Committee Collection and culture techniques of nasal lavage The collection, handling, and culturing techniques were carried out using the novel method described by Ponikau et al [5] in They found that this method allows collecting as much mucus as possible, improving the fungal yield in the. Two puffs of phenylephrine hydrochloride 1% are sprayed into each nostril to produce vasoconstriction. After approximately 2 minutes, each nostril is flushed with 20 ml of sterile saline using a sterile syringe with a sterile curved blunt needle. The patient takes a deep inspiratory breath and holds it before the injection of saline. The patient then forcefully exhales through the nose during the flushing. The return is collected in a sterile pan. The collected fluid was placed into centrifuge tubes and sent directly to the mycology laboratory where the specimen is processed under a laminar flow hood to prevent contamination. One vial (10 ml) of sterile dithiothreitol (Sputolysin; Oxoid, England) was diluted with 90 ml of sterile water. The collected specimen is suspended with an equal volume of diluted dithiothreitol and vortexed for 30 seconds. The mixture was allowed to stand at room temperature for 15 minutes while the dithiothreitol breaks apart the disulfide bonds, thus liquefying the mucus and allowing the fungal elements to be released from the viscid mucus and contact the growth media adequate for fungal growth. The mixture is then centrifuged at 3000 rpm in a 50-mL tube for 10 minutes. The supernatant was discarded, and the sediment was vortexed for 30 seconds. One-half milliliter of the prepared sediment was inoculated onto each of 3 types of : (1) Sabouraud dextrose agar (SDA) plate, (2) SDA plate containing gentamicin (5 μg/ml) and chloramphenicol (15 μg/ml), and (3) inhibitory mould agar containing 5% chloramphenicol (15 μg/ml) and cycloheximide (5 mg/ml). The plates were incubated at 30 C and allowed to grow for 30 days. The plates were examined at 2-day intervals, and all were identified. The resulting growth was subcultured on corn meal agar for proper identification and isolation of the different species harvested Collection of surgical specimens and histologic examination The patients were subjected to endoscopic sinus surgery under general anesthesia. The principle of maximum mucus preservation was adhered to during the acquisition of surgical specimens. This enabled the pathologist to find allergic mucin and fungal elements within the mucus. The use of suction devices was limited. The mucus was manually removed together with inflamed tissue and placed on a saline-moistened nonstick sheet. Specimens were not placed directly on a surgical towel or on gauze because these carriers absorb a large amount of the mucus. The paraffin-embedded blocks were cut into several serial sections of 5 mm each and baked onto positively charged glass slides. The sections were stained with hematoxylin and eosin (H&E), Gomori methenamine silver (GMS), periodic acid-schiff stain (PAS), and chitinase stain. Each of the H&E-, GMS-, and PAS-stained specimens was thoroughly examined under light microscopy for the presence of fungal elements. A specimen was considered positive if at least one fungal hypha could be clearly identified in an entire slide. The serial section slides immediately adjacent to the PAS slides were used for chitinase staining following the technique stated by Taylor et al in The specimens were deparaffinized and rehydrated using xylene, ethanol, and distilled water. Because the eosinophil-rich environment increases the background staining of fluorescein, the sections were incubated for 30 minutes in 1% chromotrope 2R to decrease nonspecific binding. The specimens were then washed in phosphate-buffered saline for 15 minutes and incubated in 5% periodic acid for 10 minutes. The slides were then rinsed with distilled water and incubated in 5% bovine serum albumin containing 0.1% azide for 10 minutes. One drop of the fluorescein-labeled chitinase stain

3 310 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) (Fungalase-F; Anomeric, Inc, Baton Rouge, LA) was then added; and the slides were incubated in a dark, humidified chamber for 30 minutes. Once the staining was completed, the slides were rinsed and washed in phosphate-buffered saline solution for 2 minutes. The specimens were then mounted with 1 drop of Vectashield mounting medium (Oxoid, Hampshire, United Kingdom), and a coverslip was applied. All stained specimens were stored in the darkness at 4 C [6]. Each slide was then examined with a Zeiss microscope equipped with Zeiss UV FL vertical illumination for epifluorescence and a fluorescein filter system (Carl Zeiss, Inc, Oberkochen, Germany) (magnification range: 100, 200, 400, and 1000) within 1 week to avoid fading. Fluorescence photomicrographs were obtained through both the immunofluorescence microscope and the confocal laser microscope. A specimen was considered positive if all of the 3 following criteria were met: (1) the element in question enhanced brightly; (2) it had the characteristic morphology of fungal hyphae; and (3) it traveled through the mucin indicating the tubular structure, not merely overlying the specimen. Because chitinase binds to the fungal cell wall only, specimens were examined for the fluorescent outline of fungal hyphae. These outlines vary in shape, ranging from circular to oblique to longitudinal depending on the angle at which the specimen was cut. The outlined rims of the fungal hyphae could be followed as it traveled through the full thickness of the specimen, ensuring that the slides were not secondarily contaminated by environmental fungi. 3. Results In this study, the age and sex distribution of cases is seen in Table 1. The average period of the patient complaint was 1.2 years, 58 patients did not have any previous surgical intervention, 22 patients had a single previous nasal operation to relief symptoms, 15 patients had 2 operations, and 5 patients had 3 or more previous operations. All patients had a broad spectrum of inflammatory mucosal thickening ranging from minimal polypoid changes (n = 32) to massive polyposis (n = 68) Mycological results The new collection and culturing method stated by Ponikau et al (1999) that was used in this study resulted in positive for fungi in 100 (100%) of the 100 Table 1 Age and sex distribution in our study Sex n Minimum age Maximum age Mean age SD Female Male Table 2 The frequency of organisms recovered from patients and controls using the SDA medium Category of cases studied No. of organisms Frequency Percentage Controls Total Patients Total consecutive patients with CRS using the SDA medium and in 50 (100%) of the control group of normal, healthy volunteers using the same medium. The using the SDA, chloramphenicol, and gentamicin medium resulted in 98 (98%) positive of the same 100 consecutive patients and 49 (98%) positive of the control group of normal, healthy volunteers using the same medium. The using the SDA and cycloheximide medium resulted in 83 (83%) positive of the same 100 consecutive patients and 41 (82%) positive of the control group of normal, healthy volunteers using the same medium. In the patients group, an average of 2.8 organisms per patient and a maximum of 5 different organisms were found on. A total of 29 different genera or species of fungi were identified. Interestingly, in the control group of normal, healthy volunteers, an average of 2.8 different organisms per volunteer also and a maximum of 4 different organisms per subject were grown. The organisms grown from the controls Table 3 The frequency of organisms recovered from cases and controls using the SDA, chloramphenicol, and gentamicin medium Category of cases studied No. of organisms Frequency Percentage Controls Total Patients Total

4 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) Table 4 The frequency of organisms recovered from patients and controls using the SDA and cycloheximide medium Category of cases studied No. of organisms Frequency Percentage Controls Total Patients Total flavus (17%) Penicillium species (14%), and A versicolor (10%), when SDA was used as the culture medium for the nasal wash of the patients in this study. Aspergillus niger was the most common organism encountered (50%), followed by Alternaria species (28%), were not markedly different than those from the CRS patients (Tables 2-4). Aspergillus niger was the most common organism encountered (51%), followed by Alternaria species (28%), A fumigatus (28%), Pseudoallescheria boydii (20%), A Table 5 Comparison of the frequency of isolation of fungi using the 3 culture media from the patients (n = 100) Fungus Patients SDA SDA, chloramphenicol, and gentamicin Acremonium Altenaria A Niger A fumigatus A flavus A versicolor A terrus A nidulans Aspergillus spp Aureobasidum Bipolaris Cabdida albicans Chaetomium Chryosporium Cladosporium Curvularia Dactylaria Dematiaceous fungus Epicocum Fusarium Mucor Nigrosporia Rhizopus Penicillium Pithomyces P boydii Stachybotrys Trichoderma Trichosporon SDA and cycloheximide Fig. 1. The microscopic morphology of fungi encountered in patients in this present study (40 objective). (A) Curvularia species. (B) Alternaria species. (C) Stachybotrys species.

5 312 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) A fumigatus (28%), P boydii (18%), A flavus (15%), Penicillium species (10%), and A versicolor (10%), when SDA, chloramphenicol, and gentamicin were used as the culture medium for the nasal wash of the patients in this study. Aspergillus niger was the most common organism encountered (46%), followed by Alternaria species (23%), P boydii (15%), A fumigatus (14%), A versicolor (6%), and Penicillium species (5%), when SDA and cycloheximide were used as the culture medium for the nasal wash of the patients in this study (Table 5, Fig. 1). A summary to the relative incidence of different types of fungi in the 3 studied cultural media in the controls is shown in Table Statistical analysis A. Comparison of the number of negative in patients vs controls according to the culture medium used: 1. Sabouraud dextrose agar Cultures were 100% positive in both patients and controls, and no statistics were computed because negative/positive is a constant. Table 6 Comparison of the frequency of isolation of fungi using the 3 culture media from the controls (n = 50) Fungus Controls SDA SDA, chloramphenicol, and gentamicin Acremonium Altenaria A niger A fumigatus A flavus A versicolor A terrus A nidulans Aspergillus spp Aureobasidum Bipolaris C albicans Chaetomium Chryosporium Cladosporium Curvularia Dactylaria Dematiaceous fungus Epicocum Fusarium Mucor Nigrosporia Rhizopus Penicillium Pithomyces P boydii Stachybotrys Trichoderma Trichosporon SDA and cycloheximide Fig. 2. A section stained by Gomori methenamine silver stain (GMS) showing many fungal hyphae within the mucin from a case of CRS with bilateral polyposis (silver stain, original magnification 400). 2. SDA, chloramphenicol, and gentamicin Statistical comparison of the negative for both patients and controls resulted in a P value = 1.00 (ie, insignificant). 3. SDA and cycloheximide The statistical comparison of the negative for both patients and controls resulted in a P value = (ie, insignificant). B. Average number of organisms in patients vs controls according to the culture medium used: 1. Sabouraud dextrose agar The average number of organisms in patients vs controls resulted in a P value =.001 (highly significant). 2. SDA, chloramphenicol, and gentamicin The average number of organisms in patients vs controls resulted in a P value =.003 (highly significant). 3. SDA and cycloheximide The average number of organisms in patients vs controls resulted in a P value = (nonsignificant). Fig. 3. Fungal hyphae detected within mucus removed from the nose showing the fruiting pattern characteristic of Aspergillus fungus from a case of CRS with bilateral polyposis (PAS 400).

6 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) Statistical comparison between results in patients vs controls: Fig. 4. Nasal polyps from a patient of CRS with bilateral polyposis with excess mucus stained intensely with the GMS stain, making identification of fungal elements difficult. C. Statistical analysis of the culture results of the commonly encountered organisms: Aspergillus niger: Statistical comparison between results in patients vs controls: 1. Sabouraud dextrose agar 51.0% (patients) vs 50.0% (controls). with a P value = SDA, chloramphenicol, and gentamicin 50.0% (patients) vs 48.0% (controls), with a P value = SDA and cycloheximide 46.0% (patients) vs 48.0% (controls), with a P value =.817 Aspergillus fumigatus: 1. Sabouraud dextrose agar 28.0% (patients) vs 28.0% (controls), with a P value = SDA, chloramphenicol, and gentamicin 26.0% (patients) vs 28.0% (controls), with a P value = SDA and cycloheximide 14.0% (patients) vs 14.0% (controls), with a P value = Alternaria species: Statistical comparison between results in patients vs controls: 1. Sabouraud dextrose agar 28.0% (patients) vs 64.0% (controls), with a P value =.000 (highly significant). 2. SDA, chloramphenicol, and gentamicin 28.0% (patients) vs 56.0% (controls), with a P value =.001 (highly significant). 3. SDA and cycloheximide 23.0% (patients) vs 50.0% (controls), with a P value =.001 (highly significant) Histopathologic results Of the 100 patients operated upon, fungal elements (hyphae, destroyed hyphae, conidia, and spores) were detected in 60 specimens using either the PAS or the Fig. 5. Section from the previous case stained by fluorescent-labeled chitinase (fungalase) showing many fungal hyphae within the mucus that were not demonstrated using the GMS or PAS stains (fungalase 1000). Fig. 6. A part of an inflamed allergic nasal polyp covered by mucussecreting columnar epithelium with excess mucus on its surface containing clusters or sheets of degenerating eosinophils and their by-products. No fungal hyphae could be detected (H&E 100).

7 314 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) Fig. 7. Section stained by fluorescent-labeled chitinase (fungalase) showing fungal hyphae that are few and small in size cut in longitudinal and cross sections within the clusters of eosinophils, ensuring the high sensitivity of the stain (fungalase 1000). GMS stains (60%) and in all of the 100 specimens using the chitinase (fungalase) stain (100%) (Figs. 2-5). The allergic mucin, containing clusters (or sheets) of degenerating eosinophils and their by-products, was found in 92 (92%) of 100 consecutive surgical cases (Fig. 6). Interestingly, in the remaining 8 cases in which the allergic mucin was absent, the eosinophils were also almost completely absent in the harvested nasal mucosa and polyps. Two of these patients also had an acute bacterial onset with neutrophil predominance. All specimens that stained negatively on PAS and GMS were noted to have smaller and fewer hyphae as identified with the fungalase staining technique (Fig. 7). Overall, although the PAS- and GMS-stained slides were positive for the identification of larger hyphae, this staining method failed to detect many of the smaller hyphae within the same specimen that could be easily visualized with the fungalase stain. 4. Discussion The origin of CRS remains elusive. Recently, the central role of fungi in this disease process has been recognized. Fungal rhinosinusitis presents in 5 clinicopathologic forms, each with distinct diagnostic criteria, treatment, and prognosis. The invasive forms are acute fulminant, and chronic and granulomatous (indolent) invasive fungal sinusitis. The noninvasive forms are fungal ball (sinus mycetoma) and AFS [7]. AFS is the most common noninvasive form of fungal rhinosinusitis. It is a highly recurrent chronic hypertrophic rhinosinusitis that can be distinguished clinically, histopathologically, and prognostically from the other forms of chronic fungal rhinosinusitis. Patients with AFS are immunocompetent, are atopic to aeroallergens including the involved fungal organism, have nasal polyps and chronic allergic rhinosinusitis, often produce nasal casts, and may occasionally present with proptosis from orbital extension of disease [8]. Based on the clinical findings in 16 patients, Bent and Kuhn [9] proposed 5 criteria for the diagnosis of AFS: (1) nasal polyposis; (2) allergic mucin; (3) computed tomographic scan findings consistent with CRS; (4) positive fungal histology or culture; and (5) type I hypersensitivity (atopy) diagnosed by history, positive skin test results, or serology. De Shazo and Swain [10] in 1995 elaborated the diagnostic criteria for AFS, which were as follows: sinusitis of one or more paranasal sinuses on x-ray film; identification of allergic mucin by rhinoscopy at time of sinus surgery or subsequently on histopathologic evaluation of material from the sinus; demonstration of fungal elements in nasal discharge or in material obtained at time of surgery by stain or culture; and absence of diabetes, previous or subsequent immunodeficiency disease, and treatment with immunosuppressive drugs. They excluded atopy as a diagnostic criteria, and their yield was only 35% positive using SDA medium for culturing the nasal discharge obtained by swaps from the nasal cavity [10]. Recently, Ponikau and his colleagues [5] were able to grow fungal in 202 (96%) of 210 patients with CRS with or without polyposis through a novel method of nasal mucus collection and a new culturing technique for fungi; they also proposed an important criterion for the diagnosis of AFS, which is the presence of the so-called allergic mucin (clusters of necrotic eosinophils in the mucus). They found the allergic mucin in 96% of 101 CRS patients undergoing endoscopic surgery by histologic examination [5]. In their study, an average of 2.7 organisms and a maximum of 8 different organisms per patient were cultured; a total of 40 different genera of fungi were identified, and 31 species have not been associated with or described in AFS before to our knowledge. Interestingly, the control group of normal, healthy volunteers (n = 14) was 100% culture positive for fungi with an average of 2.3 different organisms per volunteer and a maximum of 4 different organisms per subject. Eight genera were identified. The organisms grown from the controls were not markedly different than those from CRS patients. Of the 101 CRS patients undergoing endoscopic surgery, fungal elements (hyphae, destroyed hyphae, conidia, and spores) were found in 82 (81%) histologic specimens; and the allergic mucin was found in 97 (96%) of the 101 consecutive histologic specimens. These data indicated that the diagnostic criteria for AFS are present in the majority of patients with CRS with or without polyposis. Based on their immunologic workup, these authors also postulated that the presence of eosinophils in the allergic mucin occurs independently from a type 1

8 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) hypersensitivity and thus should be termed eosinophilic mucin. This characteristic mucin is probably the common denominator in the pathophysiology of AFS; therefore, they proposed a change in terminology from allergic fungal sinusitis to EFS [5]. A recent study was performed in Graz (Austria) to verify the Mayo clinic report and to define the prevalence of EFS in Europe using the same methods of collection and culturing techniques. In this study, positive for fungi were obtained in 84 (91.3%) of 92 of their patients with CRS, with an average of 3.2 different species per patient; 33 different genera of fungi were identified, with a range of 1 to 9 different species per patient. In 21 (91.3%) of 23 healthy control subjects, positive fungus grew, with an average of 3.1 different species per volunteer and a range of 1 to 9 different fungi per subject. Sixteen genera were identified. Of the 37 patients undergoing endoscopic surgery, fungal elements (hyphae, destroyed hyphae, conidia, and spores) were found in 28 (75.5%) histologic specimens. Eosinophilic mucin, containing clusters or sheets of necrotic eosinophils, was found in 35 (94.6%) of 37 of the histologic specimens: overall, the criteria of AFS were present in 33 (89.2%) of 37 of their patients undergoing endoscopic surgery [11]. In our present study, we applied the same collection and fungal culturing techniques in 100 CRS patients with polyposis and 50 normal, healthy Egyptian volunteers (more than double the number of the control group in each of the Mayo clinic (Ponikau et al, 1999) and Graz (Bruan et al, 2003) studies, with the following results: Fungal were 100% positive for CRS patients and the control group using the SDA medium. Fungal were 98% positive for CRS and the control group using the SDA, chloramphenicol, and gentamicin medium. Fungal were 83% positive for CRS patients and 82% positive for the control group using the SDA and cycloheximide medium. A total of 281 positive grew using the 3 culture media, showing an average of 2.8 organisms and a maximum of 5 organisms per patient. A total of 29 different genera of fungi were identified. In our control group, a total of 140 fungus-positive grew using the 3 culture media, showing an overage of 2.8 different organisms per volunteer and a maximum of 4 different organisms per subject. The organisms grown from the controls were not markedly different as regard the types from those grown from the CRS patients. In this study, fungal elements (hyphae, destroyed hyphae, conidia, and spores) were found in only 60% of histologic specimens obtained from the patients undergoing endoscopic surgery when using the GMS and PAS stains; but with the use of the fungalase stain, there was 100% positive specimens. Allergic mucin, containing clusters or sheets of degenerating eosinophils and their by-products, was found in 92 (92%) of 100 consecutive surgical specimens. A comparison of the results of this present study to both the Mayo clinic and the Graz studies is shown in Tables 7 and 8 and is discussed below: There is difference in the fungal genera that resulted from culturing the nasal wash of the AFS patients among the 3 studies; whereas in both the Mayo clinic and the Graz studies the dematiaceous fungi particularly the Alternaria species were more prevalent, in the present study, the Aspergillus species were the significantly predominant organisms. This may be due to environmental or genetic factors, as our study population was collected from the African Mediterranean population, whereas in the other 2 studies, the study population was either European or American population. Statistical analysis of the commonly encountered fungal species among our patients vs controls showed a highly significant relation with a P value of.001 for the Alternaria species, insignificant with a P value of.817 for the A niger species, and insignificant with a P value of for the A fumigatus species. The average number of organisms among our patients vs controls study using the 3 culture media was highly significant in both the SDA and the SDA, chloramphenicol, and gentamicin, with a P value of.001 Table 7 Comparison of the results of the studies of Mayo clinic and of Graz vs our present study Mayo clinic (Ponikau et al, 1999) Graz (Bruan et al, 2003) This study Patient Control Patient Control Patient Control No. of cases studied No. of positive using SDA and cycloheximide Total no. of positive using the 3 media Average no. of organisms per patient Maximum no. of organisms per patient No. of genera identified No. of positive histopathologic specimens (fungal element) 82 Not done 28 Not done 100 Not done No. of histopathologic specimens containing eosinophilic mucin 97 Not done 35 Not done 92 Not done

9 316 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) Table 8 Comparison of the results of the studies of Mayo clinic and of Graz vs our present study Fungal genera No. of positive (Mayo clinic, n = 210) No. of positive (Graz, n = 92) Acremonium Alternaria Ampelomyces species Aporospora species Arthrinium species Arachniotus citrinus Arthrographis kalrae Aspergillus A clavatus A fischerianus A flavus A fumigatus A glaucus A nidulans A niger A rubrobrunneus A tetrazonus A terreus A versicolor A versiforme Aspergillus species Aureobasidium Basidiomcota Beauveria Bipolaris Bjerkandera adusta Botrytis cinerea Candida C albicans C glabrata C krusei C lipolytica C lusitaniae C parapsilosis C tropicalis Candida species Chaetomium Chryosporium Cladosporium Cryptococcus C albidus C laurentii Cryptococcus sp Curvularia Dactylaria spp Dematiaceous fungus Echinodontium taxodi Epicoccum Exophiala jeanselmei Fusarium Geotrichum Gliomastix Heterobasidion annosum Hormonema dematioides Hypocrea schweinitzii Leptosphaeria microscopia No of positive (this study, n = 100) Table 8 (continued) Fungal genera No. of positive (Mayo clinic, n = 210) No. of positive (Graz, n = 92) Monilia Mucor Nigrospora Oidiodendron Paecilomyces Papularia Penicillium Phoma Pithomyces Pleospora papaveracea Pseudallescheria boydii Rhinocladiella Rhizopus Rhodotorula R minuta Rhodotorula sp Saccharomyces cerevisiae Sagrahamala Scolecobasidium Scopulariopsis S brumptii Schizophyllum commune Sclerotinia sclerotiorum Scopulariopsis Stachybotrys Thanatephorus cucumeris Trichaptum fusco-violaceum Trichoderma Tricholoma robustum Trichophyton T rubrum Trichophyton sp Trichosporon beigelii Ustilago 13 1 Total no. of organisms No of positive (this study, n = 100) and.003, respectively. In the SDA and cycloheximide, the P value was Analysis of the above-mentioned data of our study gives rise to the following facts: Although the Aspergillus species was more prevalent rather than any other fungal genera in both patients and controls, statistical comparison of the most commonly encountered fungal species between patients and controls proved that Alternaria species was statistically more prevalent among patients rather than controls, a matter that may raise the case against Alternaria species having a role as the causative organism in patients with AFS. There is close similarity of the number and types of organisms between both patients and controls.

10 A. Bassiouny et al. / American Journal of Otolaryngology Head and Neck Medicine and Surgery 32 (2011) However, the average number of organisms was statistically higher in patients than in controls, a matter that should draw our attention to the fact that the disease may be related to the amount of the fungal burden present in the mucus and not the mere presence of fungi. Statistical analysis of the histopathologic results in the present study directed our attention to the fact that the fungalase stain has 100% sensitivity compared with the 60% sensitivity of GMS and PAS stains in identifying fungal elements in this study, and with the 81% sensitivity in the Mayo clinic study and the 75.5% sensitivity in the Graz study. This fact conjugates fungalase staining with every AFS-suspected specimen. 5. Conclusion We have confirmed the presence of allergic mucin in 92% of our patients having CRS with polyposis by means of histologic examination of tissues removed during nasal surgery. In the same study, we have found fungal organisms in 100% of our patients using recent mycological and histologic techniques. Because the major markers of AFS are the striking numbers of eosinophils in the nasal mucus of the patients producing the allergic mucin (clusters of necrotic eosinophils in the mucus), in contrast to the near absence of eosinophils in healthy controls, our data show that the postulated criteria for the diagnosis of AFS were present in 92% of CRS with polyposis, suggesting that fungi are involved in the disease process of every CRS patient. However, to our surprise, fungal organisms were also detected in the nasal mucus of the majority of healthy control subjects; and the organisms grown from the controls were not markedly different than those from the CRS patients. However, the average number of organisms was higher in the patient group than in the controls, a matter that should draw our attention to the fact that the disease may be related to the amount of the fungal burden present in the mucus and not the mere presence of fungi, giving rise to the future role of the quantitative analysis of the fungal DNA in CRS using polymerase chain reaction. The inability to detect fungal organisms in the eosinophilic mucin in prior studies demonstrates the limitations associated with the GMS and PAS stains and that the chitinase-staining technique has greater sensitivity and specificity in detecting fungal organisms within the mucin of the patients. Eventually, our increased awareness toward the rising role of the fungal etiology implementing an allergic immunologic reaction in all cases of CRS with bilateral polyposis may lead us to a totally different concept in the management of CRS, as it would guide the surgeon on the surgical approach, the extent of resection, and any perioperative adjuvant medical therapy including immunotherapy and local antifungal therapy. 6. Conflict of interest The authors of this work as listed below confirm that they have read and approved the paper and the contribution bearing their names in it, and they state that there has been no conflict of interests and no financial support: 1. Dr Ahmed Bassiouny, MD: suggesting the idea, performing surgeries. 2. Dr Ashraf Ragab, MD: performing surgeries. 3. Dr Abd el fattah Attia, MD: mycological assistance. 4. Dr Ahmed Atef, MD: performing surgeries and writing the manuscript. 5. Dr Nadia Hafez, MD: mycological assistance. 6. 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